The proposed research involves studying the molecular mechanisms involved in the regulation of the synthesis and secretion of thyrotropin (TSH). A pituitary tumor which is induced by radiothyroidectomy in mice has been shown to be composed of a nearly homogeneous population of thyrotrophs. In short term culture, these cells synthesize and secrete TSH; TSH production in most tumors is responsive to stimulation by thyrotropin releasing hormone (TRH) and inhibition by thyroid hormones. TRH has been shown to bind to a putative receptor on the plasma membrane and may have its effect(s) mediated by cyclic nucleotides. The thyroid hormones, triiodothyronine (T3) and thyroxine (T4), may exert their effect on TSH secretion via a short-lived, intracellular polypeptide. In order to establish a more ideal cell system for this study, we will disperse, grow in long-term culture and clone pure cell lines from these tumors. The kinetics of the synthesis and secretion of TSH and its alpha and beta subunits will be defined initially in short-term cultures and then in permanent cultures of these cells. The subcellular events involved in TRH stimulation will be characterized with respect to binding to putative receptors, to possible separate effects on TSH synthesis and secretion, to stimulation of synthesis of the subunits and a possible limiting role for the beta subunit, and to interactions with thyroid hormones. The subcellular mechanisms involved in thyroid hormone inhibition will be studied with respect to their binding to putative receptors, to effects on synthesis and secretion of TSH and its subunits, and to demonstration of the intracellular mediator of inhibition of TSH secretion. It is hoped that these studies will define the molecular mechanisms involved in the regulation of synthesis and secretion of TSH and the interaction of the major hormonal mediators of this regulation, TRH, and thyroid hormones.